1. Field of the Invention
The present invention relates generally to waveguide-binding sensors for use in fluorescence assays, and, more particularly, to highly sensitive fiber-optic waveguide-binding sensors that remotely sense fluorescence radiation during assays in liquid solutions.
2. Description of the Related Art
The evanescent wave portion of an electromagnetic field produced by light propagating through an optical waveguide characteristically penetrates a few hundred nanometers into the medium surrounding the optical waveguide. This evanescent wave can excite fluorescent molecules, e.g., fluorophores, to fluoresce when they are bound by molecules near the optical waveguide surface. The application of this phenomenon to an immunoassay sensor, wherein the biological recognition (binding) of an antigen by antibodies attached to the waveguide surface with concomitant displacement of fluorescent-labeled antigen is measured as a change in fluorescence, was first disclosed in A New Immunoassay Based on Fluorescence Excitation by Internal Reflection Spectroscopy by Kronick and Little, 8 JOURNAL OF IMMUNOLOGICAL METHODS, p. 235 (1975), incorporated by reference herein in its entirety and for all purposes.
The use of optical fibers as a special class of waveguides for immunoassay sensors is also known. For example, U.S. Pat. No. 4,447,546, incorporated by reference herein in its entirety and for all purposes, discloses the use of optical fibers as waveguides which capture and conduct fluorescence radiation emitted by molecules near the optical fiber surfaces. However, conventional waveguide-binding sensors for use with assays of aqueous fluids have demonstrated inadequate sensitivity. Specifically, poor sensor performance is attributed at least in part to the small size of the sample being analyzed, typically, one or several monolayers in depth and the small surface area of the optical waveguide. These factors limit the number of fluorophores which may be excited. More serious sensor performance degradation is attributable to the effects of a weak evanescent wave which fails to excite enough fluorophores to produce detectable levels of fluorescence and inadequate coupling of the fluorescence into the waveguide for subsequent detection.
Increasing the strength of the evanescent wave penetrating into a fluid sample to be assayed increases the amount of fluorescence, thereby, increasing sensor sensitivity. Each mode (low and high order) propagating in the fiber has a portion of its power in the evanescent wave. Higher order modes have a larger percentage of their power in the evanescent wave and so make a larger contribution to power in the evanescent wave. However, these higher order modes are weakly guided, lossy, and can easily leak at a discontinuity or a bending point along the waveguide.
The use of tapered optical fibers to increase the sensitivity of fiber-optic assay systems is known. For example, U.S. Pat. Nos. 4,654,532 and 4,909,990, both incorporated by reference herein in their entirety and for all purposes, disclose the use of optical fibers as sensors used in conjunction with assays. In U.S. Pat. No. 4,654,532, an unclad, tapered optical fiber that is completely isolated from the sample fluid.
The introduction of a tapered section of the optical waveguide, however, fails to address certain important issues central to the sensitivity of these sensors, especially in remote sensing applications. In particular, the higher order modes propagating in the section of the waveguide where the fluorophores are found (the distal end) contribute the most to power in the evanescent wave and comprise the majority of the fluorescence coupled back into the fiber. These higher order modes typically propagate with greater loss than lower order modes.
For an incident beam of light of wavelength .lambda. traveling within a cladded core and intersecting the edge of a cladded core at the core and cladding boundary at an incident angle .theta. (measured from the normal of the reflecting edge) wherein the core has an index of refraction of n.sub.core and the cladding has an index of refraction of n.sub.cladding, the thickness d.sub.p of the evanescent wave region contiguous and along the outer edge of the core penetrating into the cladding is given by the formula: ##EQU1## If a portion of an optical fiber is unclad (i.e. the cladding is removed) and the bare core is surrounded by a solution having an index of refraction of n.sub.solution, then, for an incident beam of light of wavelength .lambda. traveling within an uncladded core and intersecting the edge of the uncladded core at the core and solution boundary at an incident angle .zeta. (measured from the normal of the reflecting edge) wherein the core has an index of refraction of n.sub.core and the solution has an index of refraction of n.sub.solution, the thickness d.sub.p of the evanescent wave region contiguous and along the outer edge of the core penetrating into the solution is given by the formula: ##EQU2## Typically, the evanescent wave region has a thickness d.sub.p of between about 50-500 nm depending in part on the angle of incidence .theta. as described by the equations above. Fluorescence radiation excited within the evanescent wave region coupled into the core propagates in higher-order modes and is susceptible to losses due to microbending and V-number mismatch along the optical fiber.
U.S. Pat. No. 5,061,857, to Thompson et al., entitled Waveguide-Binding Sensor for Use With Assays, filed Nov. 9, 1990 and issued Oct. 29, 1991 (the entirety of which is incorporated by reference herein for all purposes) addresses concerns about poor V-number matching and the loss of poorly guided fluorescence radiation along the length of the optical fiber. The probe is inwardly tapered from the proximal to the distal end at an angle such that the incident light beam of light traveling through the fiber does not exceed the critical angle measured from the normal of the reflecting edge to the incident beam of light. Thus, total internal reflection (TIR) of the incident beam of light traveling within the optical fiber is maintained. The fiber may also be variably doped along its surface to similarly change the V-number along the length of the fiber. Fluorescence coupled in from radii above the V-number matching radius is lossy due to the V-number mismatch.
While the approach described in U.S. Pat. No. 5,061,857 improves over the prior art, it still results in significant losses. Most of the signal in this type of fiber optic sensor is generated at the tip. If the tip is damaged, the entire probe is ruined. Also, a significant length of the tapered portion is above the V-number matching radius. Thus, most of the signal from this V-number mis-matched portion is lost.